Growth, differentiation, migration, death and the like of cells are mediated by macromolecular interactions such as protein-protein or protein-nucleic acid interactions. Signals from outside of cells pass through receptors located on the cellular membrane and are transmitted to the nucleus of a cell through various biochemical reactions, where they express specific genes. This transfer of external signals into a cell is accomplished by protein interactions of several stages. For example, growth factors or cytokines bind to corresponding cell-surface receptors. This binding induces the receptors to cluster. The clustering of receptors by ligands induces clustering of the intracellular domains of the receptors, thereby causing interactions with signaling-related proteins. Through this signaling mechanism, intermediate proteins capable of transferring signals are produced by phosphorylation by protein kinases, dephosphorylation by protein phosphatases, or the like. As a result, the signals are transmitted to transcriptional activator proteins (Helden, C. H., (1995) Cell 80, 213-223). The activated transcriptional activators bind to DNAs and interact with basal transcriptional regulator proteins such as RNA polymerases to activate specific genes. Such interactions enable transcription to occur specifically in specific tissues during embryologic processes or in response to external stimulations. Abnormal modification, inhibition or acceleration of such interactions between specific proteins, which may caused by intrusion of foreign matters, genetic modification of internal activator proteins, or the like, may be the cause of a disorder. Accordingly, there have been consistent researches because substances that can regulate the interactions may provide a way to treat the disorder.
The methods for analyzing the interactions of biomolecules, particularly the binding properties thereof, include traditional in vitro methods such as cross-linking, affinity chromatography, immunoprecipitation (IP), or the like. These methods require the production, isolation and purification of protein and are disadvantageous in that an information different from the actual interaction may be obtained depending on the buffer condition in the test tube, the secondary modification of extracted proteins, or the like.
In order to make up for these drawbacks of the in vitro methods, in-cell methods such as yeast two-hybrid (Y2H), fluorescence resonance energy transfer (FRET) and bimolecular fluorescence complementation (Bi-FC) techniques have been developed. These methods have advantages and disadvantages mentioned below.
Y2H is currently the most widely used technique along with immunoprecipitation. It is advantageous in that large-scale screening is possible using a gene library, but is disadvantageous in that investigation of membrane proteins or nuclear proteins such as transcriptase is difficult and there is a high probability of false positive. Besides, this method is inappropriate to find a substance capable of regulating protein-protein interactions. In the Y2H technique, the interaction between two proteins is detected based on the color change of colony to blue as X-gal is decomposed when β-galactosidase is expressed by the reporter gene. Since the screening technique of detecting the color change from blue back to white by a candidate substance is a negative screening, it is probable that a substance which has actually an inhibitory effect may be unnoticed. Further, since the detection itself is somewhat ambiguous, the technique is not suitable for general drug screening.
The FRET method provides good accuracy, but it is disadvantageous in that positioning of fluorescent proteins or fluorescent materials, which is required for the fluorescence resonance energy transfer to occur, is difficult, thereby having low rate of experimental success. The Bi-FC method is advantageous in that it is applicable to membrane proteins or nuclear proteins. However, like the FRET method, it is disadvantageous in that relative positioning of proteins for complementary binding is difficult, thereby having low rate of success.
Therefore, various modified methods have been proposed to overcome the disadvantages of the above-described methods. However, there is a consistent need for an effective method for detecting the binding of biomaterials. Particularly, a detecting system enabling the detection of proteins interacting with target proteins and enabling a more efficient detection of regulator materials that inhibit or promote the interactions between two proteins is urgently needed.